The present invention relates to proteases with low thermostability, genes required for producing the same and plasmids containing the gene, microorganisms carrying the plasmid, and a method for producing the protease using these microorganism. More specifically, this invention relates to a milk clotting enzyme for use in cheese production.
As the protease for cheese production, i.e., a milk clotting enzyme, calf rennet has conventionally been employed. The milk clotting activity of calf rennet is predominantly attributable to chymosin as an acid protease, and it is because of the higher specificity to milk casein and the low thermostability that chymosin is an excellent milk clotting enzyme.
With the decrease in the number of slaughtered calves, however, the stable supply of calf rennet has also been decreasing. As an alternative enzyme to calf rennet,"microbial rennets", representatively illustrated by Mucor rennet (MR) produced by Mucor pusillus (Japanese Patent Publication No. 40-18830) are therefore predominantly used as rennet.
However, many of these enzymes have a low ratio of C/P (a ratio of milk clotting activity to proteolitic activity) which is important as a milk clotting enzyme property. The result is that some of these enzymes deteriorate cheese flavor. Also, many of the enzymes generate bitter peptides during ripening because the enzymes have a relatively high thermostability, and are not inactivated during the cooking process in cheese production.
In order to solve these problems, the production of calf chymosin by introducing the gene thereof into Escherichia coli (E. coli) and the like was accomplished by using genetic engineering techniques (Japanese Patent Publication No. 62-40999).
Attempts have also been made to lower the thermostability of MR, which is the preferred microbial rennet, by chemical modification of the enzyme (Japanese Patent Publication No. 2-18834; Japanese Patent Laid-Open Publication No. 61-185186 and vice versa).
Although these methods have been somewhat successful, they are not satisfactory. Thus, a type of enzyme with a higher C/P ratio and a lower thermostability has been demanded.
Alternatively, the cloning of the MR gene has been established (Tonouchi, N., et al., Nucleic Acid Res. 14, 7557-7568(1986)), and the present inventors disclose that the gene can be expressed in yeast (Yamashita, T., et al., Mol. Gen. Genet. 210, 462-467 (1987)).
However, all of the plasmids used for the expression of the MR gene in yeast delete a part of the LEU2 gene, so the transformation efficiency thereof in yeast is not high. Also, such plasmids are inconvenient for handling. For example, it is hard to manipulate the plasmids, because the plasmids each have an unnecessary sequence downstream from the terminator and have the sequence of a whole length of 2 .mu.m DNA. In addition, these plasmids are unstable.
Therefore, a means of using yeast to stably supply milk clotting enzymes having a higher C/P ratio and a lower thermostability is desired.